The human papillomaviruses (HPV) are major etiologic agents of several human cancers and have been found in >95% of cervical cancers and a significant number of other anogenital cancers as well as oral, laryngeal, and nasal cancers. Cervical cancers are associated with a high risk subset of HPVs of which HPV-16 is the most common. The E6 and E7 viral oncogenes interfere with the functions of p53 and pRb, respectively, and thus disrupt regulation of the cell cycle and promote genomic instability. The E6 protein has also been shown to induce expression of telomerase. Progression of HPV-16 infection to high grade lesions and invasive cervical cancer requires significantly long lag periods, consistent with the accumulation of multiple genetic and/or epigenetic changes. These changes are manifested by alterations in cellular gene expression as well as key changes in the biological behavior of the cells. The goals of this project are 1) to identify changes in cellular gene expression induced by HPV infection and 2) to identify changes in cellular and papillomaviral gene expression in cervical epithelia during progression of low grade squamous intraepithelial lesions (LGSIL) to high grade SIL (HGSIL) to invasive cervical cancer. Our ultimate goals are to identify biomarkers for diagnosis and prognosis as well as to identify pathways disrupted during cervical cancer progression. This part of the project is being done as a collaboration with Dr. Sophia Wang and coworkers in DCEG, NCI as part of a large molecular epidemiological study on cervical cancer progression. A major emphasis in the past year has been the investigation of the influence of keratinocyte culture conditions on cellular senescence and on the transformation of human foreskin keratinocytes (HFKs) by HPV-16. HFKs were transduced with retroviral vectors expressing either HPV-16 E6, E7, or E6 and E7 proteins. Non-transduced and transduced HFKs were then serially passaged either in serum-free medium (KGM) on plastic or in F media on 3T3 fibroblast feeders. The expression of HPV-16 E6 and E7, hTERT and p16INK4A mRNAs was monitored by real time quantitative RT-PCR (QRT-PCR) and telomerase activity was analyzed by a very quantitative and novel real time Q-PCR-based TRAP assay developed in our lab. These studies led to several interesting observations and conclusions. Non-transduced HFKs grown on plastic lose telomerase activity within a few passages and p16 levels increase, inducing cell cycle arrest and senescence. In contrast, growth of non-transduced HFKs on feeders maintains telomerase activity and proliferative compacity for 60 population doublings. Furthermore, feeder culture conditions induce telomerase activity in HFKs previously grown on plastic. This effect of the feeder culture system is fully reversible and due predominantly to the medium rather than the fibroblasts. The HPV-16 E6 protein is known to induce telomerase activity in HFKs. We now show that the E7 protein dramatically augments the induction of telomerase activity by E6 in HFKs grown on feeders. However, this effect of E7 on telomerase activity is seen only in the presence of E6 and feeder culture conditions. Our current goal is to determine the mechanisms by which feeder culture conditions and the HPV E7 protein induce telomerase activity. These studies demonstrate that culture conditions significantly alter the host response to HPV oncogenes. To accomplish our second goal, we use Laser Capture Microdissection to obtain pure populations of normal, precancer, and invasive cervical cancer cells for expression analysis. We have previously developed signal amplification technologies that allow us to get valid microarray expression data from as few as 5,000 cells. In the past year, considerable effort has been expended to improve our abilities to work with small amounts of RNA. In addition, we are currently carrying out a pilot project with DCEG/NCI designed to identify the best methods of tissue preservation for molecular studies. Normal cervical tissues and invasive cervical cancers have been snap frozen, treated with RNALater (Ambion), or fixed in ethanol and polyester wax embedded and are currently being evaluated for RNA quality. We are also in the process of selecting a clinical site for the collection of specimens. Finally, we are continuing to develop isoform-specific real time QRT-PCR assays to complement the microarray analyses of gene expression. Some of these assays (XP-C, VEGF) are also being used in collaborative studies with other NCI laboratories.